RAD51介导的核仁封存及DNA修复通路选择在高级别卵巢浆液性癌铂类耐药中的作用研究

High grade serous ovarian cancer (HGSOC) is reported with high incidence rate, rapid progression and poor prognosis due to its elevated level of resistance to platinum-based therapies. DNA damage repair protein (RAD51) mediated homologous recombination (HR) repair pathway is actived in the formation of platinum resistance.In our early study, we have found that RAD51 was highly expressed in platinum-resistant HGSOC cells combined with an increased level of nucleolar sequestration of RAD51 protein. Based on these data we suggest there might be a DNA repair pathway selection mechanism between HR and NHEJ which could be linked with platinum resistance regulation in HGSOC. In this project the role of RAD51 nucleolus sequestration in HGSOC, as well as the possibility of p14ARF and ubiquitination involved regulation of RAD51 nucleolus sequestration would be investigated. The RAD51-mediated DNA repair pathway selection (HR and NHEJ) and key factors in the pathways regulation would be screened to explore their roles in platinum-based drug resistance. New strategy of predicting platinum resistance and increasing platinum sensitivity should be investigated by targeted inhibition of key factors in multiple pathways in HGSOC.

高级别卵巢浆液性癌以发病率高、病情进展快、预后差成为临床妇产科医生关注的焦点，其中铂类耐药现象是致其预后不良的严重制约因素，以DNA损伤修复蛋白RAD51为核心的同源重组通路（HR）的活跃构成铂类耐药的主轴。申请人前期工作发现，RAD51在高级别卵巢浆液性癌铂类耐药细胞中高表达，且耐药时将大量RAD51蛋白封存在核仁，由此推测RAD51核仁封存是否启动（或关闭）经典HR通路，并通过DNA修复通路选择机制介导其他通路（非同源末端连接NHEJ）参与卵巢癌铂类耐药调控。基于上述本项目拟明确RAD51核仁封存在高级别卵巢浆液性癌铂类耐药中的作用，深入研究p14ARF诱导RAD51核仁封存并影响其蛋白泛素化途径、RAD51介导的DNA修复（HR和NHEJ）通路选择及筛选通路中关键因子并探讨其对铂类耐药的调控机制。通过联合干预和靶向抑制多条通路中的关键因子，寻找有效预测甚至逆转卵巢癌铂类耐药的新策略。

高级别浆液性卵巢癌（HGSOC）以发病率高、病情进展快、预后差成为临床妇产科医生关注的焦点， 其中铂类耐药现象是致其预后不良的严重制约因素，以DNA损伤修复蛋白RAD51为核心的同源重组通路（HR）的活跃构成铂类耐药的主轴。前期工作发现，RAD51在高级别卵巢浆液性癌铂类耐药细胞中高表达，且耐药时可将大量RAD51蛋白封存在核仁，从而激活核内大量转录因子和经典HR通路，参与卵巢癌铂类耐药调控。本研究通过多种功能学实验和免疫荧光定位明确了RAD51 核仁封与多种 HGSOC 细胞铂类耐药的关系，并与临床HGSOC患者临床病理参数、耐药情况、复发转移进行相关性分析，最终建立起RAD51核仁封存与卵巢癌铂类耐药的关键证据。进一步应用Label-free探究HGSOC铂类敏感细胞系PEO1和铂类耐药细胞系PEO4中分别给予顺铂作用后两者的差异表达蛋白及相关富集通路，根据部分上调表达显著的差异蛋白进行平行反应监测（Parallel Reaction Monitoring，PRM）验证，最终选择RAD51 的上游调控基因RacGAP1及Hippo-YAP1通路作为后续机制研究的突破点。通过构建RacGAP1 shRNA干扰载体pLVX-shRNA-RacGAP1，结果显示：Hippo信号通路中主要的的转录调节因子YAP1在核内表达水平降低，胞浆中p-YAP1的表达水平升高，抑制核内相关转录因子活性，RAD51核仁封存程度降低，并提高了对铂类药物的敏感性。本研究通过靶向调控RacGAP1-YAP1-RAD51通路中的关键因子能够有效预测、改善甚至逆转HGSOC铂类耐药，并为卵巢癌靶向药物的研发提供新策略。